In general, the invention features fluorescent Axcex2 peptides and methods for their use.
The extracellular deposition of xcex2-amyloid in senile plaques is one of the neuropathological hallmarks of Alzheimer disease (AD). The major constitutive component of amyloid plaques is the Axcex2 peptide, a 39 to 43 residue polypeptide (Selkoe et al., (1986) J. Neurochem. 46:1820-1834) that is proteolytically derived from the amyloid precursor protein (APP), a much larger type I transmembrane protein. Axcex2 is folded into the xcex2-sheet structure that is characteristic of amyloid fibrils.
Amyloid plaque formation likely involves two basic steps: the initial formation of a seeding aggregate that establishes the amyloid fibril lattice (Kirschner et al., (1987) Proc. Natl. Acad. Sci. USA 84:6953-6957), followed by the elongation of the fibril by the sequential addition of subunits (Maggio et al., (1992) Proc. Natl. Acad. Sci. USA 89:5462-5466). Some of the key parameters that promote the assembly of amyloid fibril include high peptide concentration, long incubation time, low pH (pH 5-6) (Barrow et al., (1991) Science 253:179-182; Burdick et al., (1992) J. Biol. Chem. 267:546-554; and Fraser et al., (1991) Biophys. J. 60:1190-1201), solvent composition (Shen and Murphy, (1995) Biophisical J. 69:640-651), and salt concentration (Hilbich et al., (1991) J. Mol. Biol. 218:149-163). Assembly of Axcex2 into the fibrils may also be promoted by molecules that interact with Axcex2 and increase its rate of aggregation in vitro including ApoE (Strittmatter et al., (1993) Proc. Natl. Acad. Sci. USA 90:8098-8102; and Wisniewski and Frangione, (1992) Neurosci. Lett. 135:235-238), xcex11-antichymotrypsin (Abraham et al., (1988) Cell 52:487-501), complement C1q (Rogers et al., (1992) Proc. Natl. Acad. Sci. U.S.A. 89:10016-10020), heparin sulfate proteoglycan (Snow et al., (1988) Am. J. Pathol. 133:456-463), and zinc ions (Bush et al., (1994) J. Biol. Chem. 269:2152-12158; and Bush et al., (1994) Science 265:1464-1467).
Although many of the parameters influencing fibril assembly have been elucidated, relatively little is known about the structure of soluble Axcex2. Gel filtration analysis of Axcex2 in solution reveals the presence of multiple, discrete structures that have variously been interpreted as monomer, dimer, trimer, and higher order aggregates (Barrow et al., (1992) J. Mol. Biol. 225:1075-1093; Bush et al., (1994) J. Biol. Chem. 269:2152-12158; Hilbich et al., (1991) J. Mol. Biol. 218:149-163; Soreghan et al., (1994) J. Biol. Chem. 269:28551-28554; and Zagorski and Barrow, (1992) Biochemistry 31:5621-5631). The oligomeric structure depends on the concentration of the peptide, time of incubation, and the length of its carboxyl terminus (Soreghan et al., (1994) J. Biol. Chem. 269:28551-28554).
In general, the invention features a composition that includes an aggregating amyloid Axcex2 peptide to which is covalently bonded a fluorescent label. In one preferred embodiment, the fluorescent label is covalently bonded to a cysteine amino acid. The invention also features a method for generating such a preferred aggregating amyloid Axcex2 peptide. The method involves (a) generating an amyloid Axcex2 peptide including a cysteine amino acid substitution; (b) covalently bonding a fluorescent label to the peptide at the cysteine amino acid; and (c) determining whether the peptide is capable of aggregating with another Axcex2 peptide.
In other preferred embodiments, the wild type amyloid Axcex2 peptide is a human Axcex2 peptide; the amyloid Axcex2 peptide has the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2; the cysteine amino acid of the peptide replaces an amino acid in a wild type Axcex2 peptide and, for example, replaces an internal amino acid or a hydrophobic amino acid of the peptide. Preferred aggregating Axcex2 peptides include, without limitation, those having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except that a cysteine replaces either a phenylalanine at position 4, an aspartic acid at position 7, a glycine at position 25, or a leucine at position 34. Preferred fluorescent labels include any thiol-reactive fluorescent dyes (for example, 5-(2-iodoacetyl)amino)ethyl)aminonapthylene-1-sulfonic acid (1,5-IEDANS) or fluorescein) or any of the light-emitting moieties chosen from the group consisting of dipyrromethene boron fluoride (Bodipy), fluorescein thiosemicarbazide (FTC), sulforhodamine 101 acid chloride (Texas Red), phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4,6-diamidino-2-phenylindole (DAPI), an indopyras dye, pyrenyloxytrisulfonic acid (Cascade Blue), 514 carboxylic acid (Oregon Green), eosin, erythrosin, pyridyloxazole, benzoxadiazole, aminonapthalene, pyrene, maleimide, a coumarin, 4-fluoro-7-nitrobenofurazan (NBD), 4-amino-N-[3-(vinylsulfonyl)-phenyl]naphthalimide-3,6-disulfonate) (Lucifer Yellow), propidium iodide, a porphyrin, a cyanine dye (CY3, CY5, CY9), a lanthanide cryptate, a lanthanide chelate, or a derivative or analog thereof.
The aggregating Axcex2 peptides of the invention are useful in methods for detecting or monitoring Axcex2 production, accumulation, aggregation, or disaggregation. One particular method for the detection of an amyloid aggregate (for example, an amyloid plaque) involves (a) contacting the sample with an aggregating amyloid Axcex2 peptide to which is covalently bonded a fluorescent label; and (b) detecting the fluorescent label in association with the sample as an indication of an amyloid aggregate.
In preferred embodiments, this method is carried out to diagnose Alzheimer""s disease or a predisposition thereto; the fluorescent label is covalently bonded to a cysteine amino acid; the cysteine amino acid replaces an amino acid in a wild type Axcex2 peptide; the cysteine amino acid replaces a hydrophobic amino acid or an internal amino acid in a wild type amyloid Axcex2 peptide; the aggregating Axcex2 peptide is chosen, without limitation, from peptides having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except that a cysteine replaces either a phenylalanine at position 4, an aspartic acid at position 7, a glycine at position 25, or a leucine at position 34; and the fluorescent label is a thiol-reactive fluorescent dye (for example, 5-(2-((iodoacetyl)amino)ethyl)aminoapthylene-1-sulfonic acid (1,5-IEDANS) or fluorescein) or is chosen from the light-emitting moieties, dipyrromethene boron fluoride (Bodipy), fluorescein thiosemicarbazide (FTC), sulforhodamine 101 acid chloride (Texas Red), phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4,6-diamidino-2-phenylindole (DAPI), an indopyras dye, pyrenyloxytrisulfonic acid (Cascade Blue), 514 carboxylic acid (Oregon Green), eosin, erythrosin, pyridyloxazole, benzoxadiazole, aminonapthalene, pyrene, maleimide, a coumarin, 4-fluoro-7-nitrobenofurazan (NBD), 4-amino-N-[3-(vinylsulfonyl)-phenyl]naphthalimide-3,6-disulfonate) (Lucifer Yellow), propidium iodide, a porphyrin, a cyanine dye (CY3, CY5, CY9), a lanthanide cryptate, a lanthanide chelate, or a derivative or analog thereof.
The aggregating Axcex2 peptides of the invention also find use in screens for identifying compounds capable of affecting the aggregation of Axcex2 amyloid peptide. One particular method involves (a) providing a sample of Axcex2 amyloid peptide; (b) contacting the sample with (i) an aggregating amyloid Axcex2 peptide to which is covalently bonded a fluorescent label; and (ii) a candidate compound; and (c) measuring association of the fluorescent label with the sample, a change in the level of fluorescent label found in association with the sample relative to a control sample lacking the candidate compound being an indication that the compound is capable of affecting Axcex2 amyloid peptide aggregation. In a preferred embodiment of this method, the sample includes unlabeled Axcex2 amyloid peptide bound to a solid support, and the aggregation is measured by association of the fluorescent label with the solid support.
The invention also includes a second exemplary method for identifying a compound capable of affecting the aggregation of Axcex2 amyloid peptide. This method involves (a) providing a sample of an aggregating amyloid Axcex2 peptide to which is covalently bonded a fluorescent label; (b) contacting the sample with a candidate compound; and (c) measuring the ability of the peptide to aggregate, a change in the amount of aggregated peptide in the presence of the candidate compound relative to a sample lacking the compound being an indication that the compound is capable of affecting Axcex2 amyloid peptide aggregation.
In preferred embodiments of both of the above screening methods, the fluorescent label is covalently bonded to a cysteine amino acid; the cysteine amino acid replaces an amino acid in a wild type Axcex2 peptide; the cysteine amino acid replaces a hydrophobic amino acid or an internal amino acid in a wild type amyloid Axcex2 peptide; the aggregating Axcex2 peptide is chosen, without limitation, from peptides having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, except that a cysteine replaces either a phenylalanine at position 4, an aspartic acid at position 7, a glycine at position 25, or a leucine at position 34; the fluorescent label is a thiol-reactive fluorescent dye (for example, -5-(2-((iodoacetyl)amino)ethyl)aminonapthylene-1-sulfonic or fluorescein) acid (1,5-IEDANS) or fluorescein) or is chosen from the group of light-emitting moieties consisting of dipyrromethene boron fluoride (Bodipy), fluorescein thiosemicarbazide (FTC), sulforhodamine 101 acid chloride (Texas Red), phycoerythrin, rhodamine, carboxytetramethylrhodamine, 4,6-diamidino-2-phenylindole (DAPI), an indopyras dye, pyrenyloxytrisulfonic acid (Cascade Blue), 514 carboxylic acid (Oregon Green), eosin, erythrosin, pyridyloxazole, benzoxadiazole, aminonapthalene, pyrene, maleimide, a coumarin, 4-fluoro-7-nitrobenofurazan (NBD), 4-amino-N-[3-(vinylsulfonyl)-phenyl]naphthalimide-3,6-disulfonate) (Lucifer Yellow), propidium iodide, a porphyrin, a cyanine dye (CY3, CY5, CY9), a lanthanide cryptate, a lanthanide chelate, or a derivative or analog thereof; and aggregation is measured by centrifugation, gel filtration, or fluorescence resonance energy transfer (FRET) analysis.
As used herein, by an xe2x80x9camyloid Axcex2 peptidexe2x80x9d is meant any xcex2-amyloid peptide or fragment thereof which aggregates under physiological conditions (for example, as tested herein).
By a xe2x80x9cwild typexe2x80x9d Axcex2 peptide is meant any naturally occurring xcex2-amyloid peptide.
By an xe2x80x9caggregatingxe2x80x9d amyloid Axcex2 peptide is meant that, under physiological conditions, the peptide (which is fluorescently labeled) exhibits at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% of the aggregate formation exhibited by a corresponding unlabeled peptide under identical conditions.
By an xe2x80x9cinternal amino acidxe2x80x9d is meant any amino acid of a peptide except the amino-terminal or carboxy-terminal residues.
The present invention provides a number of advantages. Most notably, because the peptides described herein represent the first examples of fluorescently labeled Axcex2 peptides that exhibit wild type aggregation properties, this invention enables any number of diagnostic techniques that appropriately monitor amyloid aggregation or disaggregation. In addition, also because of the peptides"" wild type aggregation characteristics, the invention enables, for the first time, screening techniques using biologically relevant fluorescent Axcex2 peptides for the discovery of compounds that affect amyloid peptide aggregation. Such compounds provide important candidate therapeutics, for example, for the treatment or amelioration of Alzheimer""s disease or its symptoms.
Other features and advantages of the claimed invention will be apparent from the following detailed description thereof, and from the claims.
The drawings will first briefly be described.